Plants made insect resistant by transformation with a nucleic acid encoding a modified Cry1Ab protein and methods for making same

ABSTRACT

The present invention relates to a DNA sequence encoding a modified Cry1Ab protein that has insecticidal activity. The invention further relates to a method for producing insect resistant plants by introducing into the genome of the plants a foreign DNA comprising such a modified cry1Ab coding sequence. The invention further relates to plants or parts thereof comprising in their genome the modified cry1Ab coding sequence of the present invention.

CONTINUING APPLICATION DATA

This application claims benefit of provisional Application No. 60/509,067, filed 3 May 2002, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a DNA sequence encoding a modified Cry1Ab protein that has insecticidal activity. The invention further relates to a method for producing insect resistant plants by introducing into the genome of the plants a foreign DNA comprising such a modified cry1Ab coding sequence. The invention further relates to plants or parts thereof comprising in their genome the modified cry1Ab coding sequence of the present invention.

BACKGROUND ART

The Gram-positive soil bacterium Bacillus thuringiensis is well known for its production of proteins or delta-endotoxins, that are toxic to a variety of lepidopteran, coleopteran, and dipteran larvae. Different strains of B. thuringiensis have been shown to produce different insecticidal crystal proteins, which are specifically toxic to certain species of insects (reviewed by Höfte and Whiteley, 1989; Schnepf et al., 1998).

The specific toxicity of insecticidal toxins produced by B. thuringiensis for target insects and their non-toxicity to plants and other organisms has made compositions comprising different Bt strains the product of choice for the biological control of agricultural insect pests. Various of the genes encoding the crystal proteins have been cloned and their DNA sequences determined (Höfte and Whiteley, 1989; Crickmore et al., 1995). This has led to the engineering of modified delta-endotoxin encoding genes and the development of plants expressing these delta-endotoxin genes to make them insect resistant.

The family of cry1 genes encode the Cry1 crystal proteins, which are primarily active against lepidopteran pests. The protoxin form of Cry1 delta-endotoxins comprises a C-terminal protoxin part, which is not toxic and is thought to be important for crystal formation (Arvidson et al., 1989). The amino-half of the protoxin comprises the active toxin segment of the Cry1 protein. Different domains have further been identified in the active toxin, which are implied in different aspects of the toxicity effect (Grochulski et al., 1995). However, these functions seem to be dependent on the delta endotoxin examined.

Significant effort has gone into modifying the cry1 genes to improve expression levels in plants while at least retaining their toxicity to the target insects. Modification of the cry1Ab and cry1Ac genes to remove putative plant polyadenylation signals and instability motifs (without altering the encoded amino acid sequences) resulted in increased resistance of the plants transformed with these sequences (van der Salm et al., 1994). Modifications of the cry1Ab gene have been described in U.S. Pat. No. 6,320,100, U.S. Pat. No. 6,180,774 and U.S. Pat. No. 6,114,608. U.S. Pat. No. 5,500,365 describes how modification in the 240 region of the coding region of a cry1Ab gene so as to remove putative plant signals is of significant importance to increase expression levels and thereby toxicity of the Cry toxin in plants.

The present invention relates to a novel modified Cry1Ab protein and DNA sequences encoding this protein, which can be used to engineer insect resistance in plants. More particularly, it was found that this modified sequence, despite having a native 240 region, ensures sufficiently high expression in plant cells to confer insect resistance to the plant or plant tissue in which it is expressed.

SUMMARY OF THE INVENTION

The present invention relates to a modified cry1Ab coding sequence, which encodes the modified Cry1Ab protein of SEQ ID NO: 1, which is an insecticidal protein. According to a particular embodiment of the invention, the DNA sequence encoding the modified Cry1Ab sequence corresponds to the sequence of SEQ ID NO: 2, or comprises the sequence of SEQ ID NO:2.

The invention further relates to chimeric genes comprising the modified cry1Ab DNA sequence of the present invention under the control of a plant-expressible promoter. According to a particular embodiment of the present invention the plant-expressible promoter is either a constitutive promoter, a tissue-specific promoter or a wound-inducible promoter or a promoter that ensures expression of the modified Cry1Ab protein at least in the cells or tissues of a plant which are susceptible to insect attack.

The invention further relates to recombinant vectors comprising the chimeric genes of the invention and to the production of transgenic plants using these recombinant vectors.

The invention further relates to plants and cells, seeds or tissues thereof, comprising in their genome a foreign DNA comprising the modified cry1Ab DNA sequence of the present invention under the control of a plant-expressible promoter.

The invention also relates to a method for engineering insect resistance in plants, by introducing, into the genome of the plant, a foreign DNA comprising the modified cry1Ab coding sequence of the present invention under the control of a plant-expressible promoter.

According to a particular embodiment of the present invention, the modified cry1Ab coding sequence is particularly suited for engineering insect resistance in agricultural crops such as corn and cotton. Most particularly, expression of the modified Cry1Ab protein confers resistance to lepidopteran pests of these plants. More particularly, these pests include, but are not limited to, major lepidopteran pests of corn, cotton and rice, such as Ostrinia nubilalis (European corn borer or ECB), Sesamia nonagrioides (Mediterranean Stalk borer), Sesamia inferens (Pink stemborer), Helicoverpa zea (corn earworm, cotton bollworm), Helicoverpa armigera (American bollworm), Heliothis virescens (Tobacco budworm), Scirpophaga incertulas (Yellow stemborer), and Cnaphalocrocis medinalis (Rice leaf folder).

DETAILED DESCRIPTION

The term “gene” as used herein refers to any DNA sequence comprising several operably linked DNA fragments such as a promoter region, a 5′ untranslated region (the 5′UTR), a coding region, and an untranslated 3′ region (3′UTR) comprising a polyadenylation site. A gene may include additional DNA fragments such as, for example, introns. While a promoter and a coding region are required in a gene used for plant transformation in the current invention, the 3′ UTR comprising a polyadenylation site need not be present in the transferred gene itself, but can be recovered in the upstream plant DNA sequences after insertion of a gene not containing a 3′ UTR comprising a polyadenylation site.

The term “chimeric” when referring to a gene or DNA sequence is used to refer to the fact that the gene or DNA sequence comprises at least two functionally relevant DNA fragments (such as promoter, 5′UTR, coding region, 3′UTR, intron) that are not naturally associated with each other and/or originate, for example, from different sources. “Foreign” referring to a gene or DNA sequence with respect to a plant species is used to indicate that the gene or DNA sequence is not naturally found in that plant, or is not naturally found in that genetic locus in that plant. The term “foreign DNA” will be used herein to refer to a DNA sequence as it has incorporated into the genome of a plant as a result of transformation.

A genome of a plant, plant tissue or plant cell, as used herein, refers to any genetic material in the plant, plant tissue or plant cell, and includes both the nuclear and the plastid and mitochondrial genome.

A “fragment” or “truncation” of a DNA molecule or protein sequence as used herein refers to a portion of the original DNA or protein sequence (i.e., nucleic acid or amino acid) referred to or a synthetic version thereof (such as a sequence which is adapted for optimal expression in plants), which can vary in length but which is sufficient to ensure the (encoded) protein is an insect toxin. A “variant” of a sequence is used herein to indicate a DNA molecule or protein of which the sequence (nucleic or amino acid) is essentially identical to the sequence to which the term refers.

Sequences which are “essentially identical” are similar to such a degree that when two sequences are aligned, the percent sequence identity, i.e. the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the sequences, is higher than 70%, higher than 85%, higher than 90%, higher than 95%, or is between 96% and 100%. The alignment of two nucleotide sequences is performed by the algorithm as described by Wilbur and Lipmann (1983) using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4.

A ‘plant-expressible promoter’ as used herein refers to a promoter that ensures expression of a coding sequence to which it is linked in a plant cell. Examples of such promoters are well known in the art. A plant-expressible promoter can be a constitutive promoter. Examples of promoters directing constitutive expression in plants are known in the art and include the 35S promoter from Cauliflower Mosaic virus, the nopaline synthase (NOS) promoter, the ubi promoter (Christensen et al. 1992), the promoter of the GOS2 gene from rice (de Pater et al., 1992). Alternatively, a plant-expressible promoter can be a tissue-specific promoter, i.e., a promoter directing a higher level of expression of a coding sequence (as can be measured by conventional RNA assays) in some tissues of the plant, e.g. in green tissues (such as the promoter of the PEP carboxylase) than in other tissues of the plant. Alternatively, a plant-expressible promoter can be a wound-inducible promoter. A ‘wound-inducible’ promoter or a promoter directing an expression pattern that is wound-inducible as used herein means that upon wounding of the plant, either mechanically or by insect feeding, expression of the coding sequence under control of the promoter is significantly increased. Examples of wound-inducible promoters include the proteinase inhibitor gene of potato and tomato (pin1 and pin2)(Johnson et al., 1989) and the promoter of the maize proteinase inhibitor (MPI) gene (Cordero et al. 1994).

The “TR2′ promoter” as used herein relates to any promoter comprising the TR2′ (or mannopine synthase, abbreviated as mas) functional part of the TR1′–TR2′ dual promoter element from Agrobacterium (Velten et al. 1984; Langridge et al. 1989). Thus, the “TR2′ promoter” can comprise the TR2′ element either alone or in combination with all or part of the divergent TR1′ element (Guevara-Garcia et al., 1998) or other (regulatory) elements. Such elements include, but are not limited to, enhancer regions, introns and the like, as long as the wound-induction characteristics in monocots, particularly corn, in accordance with the present invention are substantially retained. In one specific embodiment of this invention, transcription is directed from the TR2′ promoter region (and the coding sequence is hence downstream of the TR2′ promoter sequence), even if the TR1′–TR2′ dual promoter (or any part thereof retaining the TR2′ promoter element) is used.

‘Insecticidal’ is used herein to mean toxic to insects that are crop pests. More particularly, in the context of the present invention target insects are pests such as, but not limited to, major lepidopteran pests, such as Ostrinia nubilalis (European corn borer or ECB), Sesamia nonagrioides (Mediterranean Stalk borer), Helicoverpa zea (corn earworm, cotton bollworm), Helicoverpa armigera (American bollworm) and Heliothis viriscens (Tobacco budworm).

In one embodiment of the present invention, the DNA encoding an insecticidal crystal protein (ICP) is a modified cry1Ab DNA sequence encoding an ICP that is a modified Cry1Ab protein. The modified cry1Ab coding sequence may encode, for example, the modified Cry1Ab protein corresponding to the sequence of SEQ ID NO: 1. In an alternative embodiment of the invention the modified cry1Ab coding sequence corresponds to the sequence of SEQ ID NO: 2. The DNA sequence used for transformation of plants, particularly corn, cotton or rice, in accordance with the current invention, can also comprise other elements besides a coding region encoding the modified Cry1Ab protein of the invention. Such other elements may include a coding region encoding a transit peptide, a coding region encoding a selectable marker protein or a protein conferring resistance to a herbicide.

In one embodiment of the invention, the modified Cry1Ab protein is toxic to major lepidopteran pests of crops such as corn, cotton and rice. Plants according to the present invention, comprising a foreign DNA in their genome comprising a DNA encoding a modified Cry1Ab protein are protected against these pests, by expressing a controlling amount of this protein. The term “controlling” encompasses toxic (lethal) and combative (sublethal) amounts of Cry1Ab protein. At the same time, the plants according to the present invention are morphologically normal and may be cultivated in a usual manner for consumption and/or production of products. Furthermore, said plants substantially obviate the need for chemical or biological insecticides (to insects targeted by the modified Cry1Ab protein).

The expression level of an ICP in plant material can be determined in a number of ways described in the art. For example, expression can be measured by quantification of the mRNA encoding the insecticidal protein produced in the tissue using specific primers (such as described by Cornelissen & Vandewiele, 1989) or direct specific detection of the amount of insecticidal protein produced, e.g., by immunological detection methods. Alternatively, the expression level of a modified Cry1Ab protein according to the present invention may be represented as the percentage of soluble insecticidal protein as determined by immunospecific ELISA (for example, as described herein) related to the total amount of soluble protein (as determined, e.g., by Bradford analysis (Bradford, 1976)). An exemplary ELISA for use in measuring expression levels of ICP is a sandwich ELISA (Clark et al., 1986).

Different assays can be used to measure the insecticidal effect or efficacy of ICP expression in the plant. As noted above, the target insects of the ICP of the present invention are the major lepidopteran pests of agricultural crops such as corn, cotton and rice. Such target insects include, but are not limited to, the European Corn Borer (ECB) and Sesamia nonagrioides (SMG) in corn, the cotton bollworm (CBW) and tobacco budworm (TBW) in cotton, and the yellow stem borer, the pink stem borer and the rice leaf folder in rice. The toxicity of an ICP produced in a corn plant on ECB can be assayed in vitro by testing protein extracted from the plant in feeding bioassays with ECB larvae, or by scoring mortality of larvae distributed on leaf material of transformed plants in a petri dish (both assays as described by Jansens et al., 1997), or on plants isolated in individual cylinders. In the field first brood European corn borer (ECB1) infestation is evaluated based on leaf damage ratings (Guthrie, 1989) while evaluation of the total number of stalk tunnels per plant and stalk tunnel length are indicative of second brood (ECB2) stalk feeding damage.

Efficacy of the ICP produced in cotton plants transformed with a modified cry1Ab gene can also be measured using in vitro and/or in vivo assays. Toxicity of the transformed plant tissue to CBW larvae can be measured by feeding CBW larvae on squares, leaves or terminals and assaying weight of surviving larvae. In the field, plants are artificially infested with neonate CBW larvae and rating damage at regular intervals to leaves, terminals, squares, white bloom, and bolls (as described herein). It will be understood that similar assays can be developed for any target or non-target insect in order to determine efficacy of the ICP produced in the plant against such insect.

The plants of the present invention optionally also comprise in their genome a gene encoding herbicide resistance. The herbicide resistance gene may be the bar or the pat gene, which confers glufosinate tolerance to the plant, i.e. the plants are tolerant to the herbicide Liberty™. Tolerance to Liberty™. can be tested in different ways. For instance, tolerance can be tested by Liberty™ spray application. Spray treatments should be made between the plant stages V2 and V6 for best results (corn stages are as determined in ‘How a Corn Plant Develops’, Special Report No. 48, Iowa State University of Science and Technology, Cooperative Extension Service, Ames, Iowa, Reprinted June 1993). Tolerant plants are characterized by the fact that spraying of the plants with at least 200 grams active ingredient/hectare (g.a.i./ha), or 400 g.a.i./ha, and possibly up to 1600 g.a.i./ha (4×the normal field rate), does not kill the plants. A broadcast application should be applied at a rate of 28–34 oz Liberty™+3 lbs Ammonium sulfate per acre. It is best to apply at a volume of 20 gallons of water per acre using a flat fan type nozzle while being careful not to direct spray applications directly into the whorl of the plants to avoid surfactant burn on the leaves. The herbicide effect should appear within 48 hours and be clearly visible within 5–7 days.

Examples of other herbicide resistance genes are genes encoding resistance to phenmedipham (such as the pmph gene, U.S. Pat. No. 5,347,047; U.S. Pat. No. 5,543,306); genes encoding resistance to glyphosate (such as the EPSPS genes, U.S. Pat. No. 5,510,471); genes encoding bromoxynyl resistance (such as described in U.S. Pat. No. 4,810,648); genes encoding resistance to sulfonylurea (such as described in EPA 0 360 750); genes encoding resistance to the herbicide dalapon (such as described in WO 99/27116); genes encoding resistance to cyanamide (such as described in WO 98/48023 and WO 98/56238); and genes encoding resistance to glutamine synthetase inhibitors, such as PPT (such as described in EP-A-0 242 236, EP-A-0 242 246, EP-A-0 257 542).

Introduction of a foreign DNA into a plant cell can be obtained by conventional transformation methods described in the art. Such methods include, but are not limited to, Agrobacterium mediated transformation (U.S. Pat. No. 6,074,877, Hiei et al., 1997); microprojectile bombardment (as described, for example by Chen et al., 1994; Casas et al., 1995; Christou, 1997, Finer et al., 1999, Vasil et al. 1999); direct DNA uptake into protoplasts (as described, for example by De Block et al. 1989; Poulsen, 1996, Datta et al., 1999); electroporation (D'Halluin et al., 1992, U.S. Pat. No. 5,641,665; Bates, 1995); silicon whisker mediated DNA introduction (Dunwell, 1999); or other methods as generally reviewed by Potrykus (1990), Sawahel et al. (1995), Komari et al. (1998), Bogorad (2000) and Newell (2000).

The following non-limiting examples describe the development of a DNA sequence encoding a modified Cry1Ab protein, the construction of chimeric genes comprising this sequence for expression in plants, and insect resistant plants obtained therewith. Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA; and in Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK. Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR—Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

Throughout the description and Examples, reference is made to the following sequences represented in the sequence listing:

SEQ ID NO: 1: Modified Cry1Ab protein

SEQ ID NO: 2: DNA sequence encoding a modified Cry1Ab protein

EXAMPLES Development of a Modified Cry1Ab Gene

The modified Cry1Ab DNA sequence used herein encodes part of the Cry1Ab5 protein described by Hofte et al. (1986) corresponding to amino acid 1 to 616, which has an insertion of an alanine codon (GCT) 3′ of the ATG start codon (AlaAsp2 . . . Asp616). The protein sequence of the modified Cry1Ab protein is provided in SEQ ID NO: 1. The sequence of the DNA encoding such a modified Cry1Ab protein is provided in SEQ ID NO: 2.

Cry1Ab Gene in Corn

Development of Cry1Ab Events in Corn

For Agrobacterium transformation of corn, constructs were developed wherein the cry1Ab coding sequence was placed under the control of different promoters: 35S promoter (Odell et al. 1985), the ubi promoter (Christensen et al. 1992), the promoter of the GOS2 gene from rice (de Pater et al., 1992) with the cab22 leader from Petunia (Harpster et al. 1988), the 5′ leader sequence of the GOS2 gene from rice, containing the second exon, the first intron and the first exon of the GOS transcript (de Pater et al., 1992), or a TR2′ promoter region (Velten et al. 1984); all constructs included the 35S-bar gene. Agrobacterium-mediated transformation was done by co-cultivation of type I callus derived from immature embryo's with strain C58C1 (pTiEHAE101)(pTTS35)(Agrobacterium C58C1RifR strain cured for pTiC58 harboring the non-oncogenic Ti plasmid pTiEHA101 (Hood et al., 1986) and the plasmids in the table below containing the genes of interest placed between the T-DNA borders).

Protoplast transformation was done by PEG mediated transfection of protoplasts prepared from suspension cultures derived from Pa91xH99xHE89 Z15 embryos. The DNA used for transfection was a purified fragment of the plasmids in the table below containing the genes of interest between T-DNA borders.

Construct description Abbreviation Agrobacterium transformation PTSVH0203 P35S2-GE1-modcry1Ab-3′ocs<>p35S3-bar-3′nos p35S-cry1Ab PTSVH0207 Pubi1-ubi leader with intron-modcry1Ab-3′ocs<>p35S3-bar-3′nos pUbi-cry1Ab PTSVH0208 Pgos2-cab22 leader-modcry1Ab-3′ocs<>p35S3-bar-3′nos pGos-cab-cry1Ab PTSVH0209 Pgos2-gos leader with intron-modcry1Ab-3′ocs<>p35S3-bar-3′nos pGos-gos-cry1Ab PTSVH0212 3′nos-bar-p35S3><Tr2-modcry1Ab-3′ocs pTR2-cry1Ab Protoplast transformation PSVH0211 Pubi1-ubi leader with intron-cry1Ab53-3′ocs<>p35S3-bar-3′nos pUbi-cry1Ab PSVH0213 Pgos2-gos leader with intron-modcry1Ab-3′ocs<>p35S3-bar-3′nos pGos-gos-cry1Ab

Regenerated plantlets were selected based on Liberty tolerance and/or measurement of PAT protein levels by ELISA (Clark et al, 1986).

Evaluation of Events

General Characterization

The Agrobacterium transformants were checked for presence of vector sequence at the left border of the T-DNA. Southern blot analyses were performed with leaf material of the primary transformants (T0).

Expression of Modified Cry1Ab Protein

The events were analyzed in the greenhouse for Cry1Ab expression by detecting soluble modified Cry1Ab protein levels in different tissues by a Cry1Ab sandwich ELISA with a polycondensated IgG fraction of a polyclonal rabbit antiserum against Cry1Ab as first antibody and a monoclonal antibody against Cry1Ab as second antibody.

The expression of modified Cry1Ab protein in different tissues of early whorl plants transformed with the p35S-cry1Ab construct is provided in Table 1. Expression in the different progeny plants of one event obtained with p35S-cry1Ab event was found to be constitutive and above 0.5% on average.

TABLE 1 Expression of Cry1Ab in different tissues of the early whorl plant Event Plant no Leaf blade Root CP048-2602 0042 0.73 1.4  (construct ptsvh0203) 0032 1.12 3.18 0033 1.14 2.39 0034 1.12 2.65 0035 0.94 3.51 0039 0.88 2.48 0040 0.68 1.7  0036 0.27 0.81 0037 0.13 0.66 0038 0.15 1.15 0041 0.13 0.49 0043 0.08 0.78 0044 0.13 0.98 0045 0.08 0.55 0031 0.11 0.57 Average (SD) 0.51 (0.44) 1.55 (1.03)

The expression levels of Cry1Ab protein in different tissues in early V, R1 and R4-5 stage leaves, R4-5 stage kernels and in pollen for plants transformed with the p35S-cry1Ab, pGos-cab-cry1Ab and pTR2-cry1Ab constructs are provided in Table 2. Results are presented as averages of leaf and kernel samples taken from five plants and as averages of pollen samples taken from three plants (standard deviation in brackets). ICP expression for the one p35S-cry1Ab plant is above 0.1% total soluble protein. The pGos-cab-cry1Ab plants showed around 0.01–0.06% Cry1Ab expression in leaves. The events obtained with the pGos-gos-cry1Ab constructs showed high expression (0.2–0.6% protein) in leaf tissue. Basal expression of protein in leaves of plants transformed with the modified cry1Ab DNA sequence under control of the wound-inducible TR2′ promoter (pTR2′-cry1Ab) was low to undetectable.

TABLE 2 Expression of Cry1Ab protein in different tissues of plants transformed with the Cry1Ab gene Cry1Ab in % soluble protein/total protein 5 plants 5 plants 3 plants 5 plants 5 plants Event Construct early V/leaf R1/leaf pollen R4–5/leaf R4–5/kernel CE048-2602 p35S-cry1Ab 0.186 (0.066) 0.54 (0.38) 0 0.41 (0.17) 0.048 (0.039) CE104-0202 pGos-cab-cry1Ab  0.014 (0.0055) 0.036 (0.015) CE1014-0402 pGos-cab-cry1Ab 0.032 (0.016) 0.041 (0.011) CE122-0806 pGos-cab-cry1Ab 0.024 (0.015)  0.10 (0.013) 0 0.058 (0.036) 0 (0) CE2162-0402 pGos-gos-cry1Ab 1.27 (0.82) CE2168-0202 pGos-gos-cry1Ab 0.467 (0.23)  CE2168-1218 pGos-gos-cry1Ab  0.26 (0.026) CE21612-1402 pGos-gos-cry1Ab 0.158 (0.067) 0.256 (0.106) 0 0.153 (0.023) 0.017 (0.006) CE21614-0604 pGos-gos-cry1Ab 0.682 (0.50)  CE21614-0816 pGos-gos-cry1Ab 0.327 (0.27)   0.925 (0.198), 0.036 (0.0115)  0.44 (0.078) 0.0417 (0.021)  1 plant 0.06 CP21614-1606 pGos-gos-cry1Ab  0.38 (0.153) WI600-0218 pTR2-cry1Ab 0 0 0 0 0.002 WI600-0802 pTR2-cry1Ab 0 0 0 0.0048 (0.0016) 0.01 

In a greenhouse study, expression of modified Cry1Ab protein was determined in leaf samples of plants in V3 stage, and, where available, leaf and pollen of plants in R1 stage, and leaf, stalk and pollen of plants at harvest, for events obtained with the different constructs. Again, the plants with the pGos-gos-cry1Ab constructs showed high Cry1Ab expression in leaves (>0.2% total soluble protein) (irrespective of transformation method used). For these plants, high expression levels were also found in the stalk (0.8% total soluble protein or more on average). Events obtained from transformation with the pTR2′-cry1Ab constructs showed low (0.02% total soluble protein or less on average) or undetectable expression of Cry1Ab protein in all tissues tested.

Inducible Cry1Ab Expression

For the events obtained with the pTR2′-cry1Ab construct, studies were performed in the greenhouse to determine the expression of the Cry1Ab protein upon mechanical damage. Leaf samples were taken before wounding, and 18 h after mechanical damage and Cry1Ab protein levels were measured by ELISA (Table 3). Damaged leaf parts were excised and incubated for 18 hours in a petri dish on filter paper moistened with Murashige and Skoog (MS) medium (Plant Molecular Biology Labfax (1993) by R. D. D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK) at room temperature and then Cry1Ab protein level was measured using a sandwich ELISA assay. Unwounded control leaf parts (of similar size as the wounded leaf parts) were excised from the plants and were immediately put on dry ice for protein analysis.

TABLE 3 Expression of insecticidal protein in different plant parts before and after mechanical wounding Cry1Ab in % soluble protein/total protein V4 stage Flowering Event Leaf Leaf Root Harvest (construct) Leaf induced Leaf induced root induced Pollen Leaf Stalk Kernel WI600-0218 Mean 0.000 0.020 0.000 0.020 0.023 0.036 0.000 0.000 0.000 0.000 (pTR2′-Cry1Ab) st.dev. 0.000 0.008 0.000 0.007 0.008 0.011 0.000 0.000 0.000 0.000 WI606-0406 Mean 0.005 0.046 0.000 0.007 0.012 0.008 0.000 \ 0.002 0.002 (pTR2′-Cry1Ab) st.dev. 0.007 0.005 0.000 0.006 0.009 0.009 0.000 0.004 WI604-1602 Mean 0.002 0.104 0.001 0.020 0.020 0.024 0.000 0.004 0.004 0.001 (pTR2′-Cry1Ab) st.dev. 0.003 0.012 0.000 0.009 0.008 0.008 0.000 0.002 0.003 0.000 WI606-0802 Mean 0.003 0.064 0.001 0.010 0.027 0.037 0.000 0.002 0.004 0.003 (pTR2′-Cry1Ab) st.dev. 0.004 0.000 0.009 0.012 0.020 0.000 0.001 0.003 0.001 WI606-1206 Mean 0.001 0.081 0.000 0.022 0.032 0.024 0.000 0.004 0.002 0.001 (pTR2′-Cry1Ab) st.dev. 0.016 0.000 0.010 0.005 0.011 0.000 0.003 0.001 0.001 CE048-2402 Mean 0.83 0.614 0.280 0.397 0.196 0.253 0.000 0.376 1.120 0.047 (p35S-Cry1Ab) st.dev. 0.108 0.111 0.027 0.133 0.083 0.000 0.098 0.175 0.014 CE048-2602 Mean 0.636 0.527 0.007 0.006 0.087 0.045 0.000 0.106 0.040 0.015 (p35S-Cry1Ab) st.dev. 0.16 0.046 0.002 0.002 0.044 0.007 0.000 0.011 0.013 0.004 Control 1 Mean 0.000 0.000 0 0 0 0 0 0 0.001 0 (untransformed) st.dev. 0.000 0.000 0 0 0 0 0 0 0.002 0 Control 2 Mean 0.000 0.000 0 0 0 0 0 0 0 0 (untransformed) st.dev. 0.000 0.000 0 0 0 0 0 0 0 0 CE0104-0202 Mean 0.022 0.023 0.008 0.017 0.023 0.016 0.0001 \ \ \ (pGos/cab- st.dev. 0.009 0.005 0.005 0.004 0.012 0.005 0.000 Cry1Ab) CE1014-0402 Mean 0.025 0.017 0.007 0.010 0.028 0.034 0.000 0.010 0.040 0.002 (pGos/cab- 0.005 0.003 0.001 0.003 0.017 0.013 0.000 0.003 0.020 0.001 Cry1Ab)

Expression of the Cry1Ab protein was either absent or around the detection limit in leaves, stalk and kernels of the different pTR2′ events tested. No expression above background levels (as found in untransformed control plants) was found in leaves and pollen in whorl and pollen shedding plant stage. Constitutive expression was seen in the roots. When leaves are mechanically damaged, expression of the modified Cry1Ab protein is induced and goes up to 0.02–0.1% in V4 stage leaves.

ECB Efficacy

ECB efficacy trials were performed in the greenhouse and at two different locations in the field. At the same time, plants were evaluated for phytotoxicity effects of the constructs introduced. Table 4 shows the results of efficacy tested for ECB. In the greenhouse, ECB efficacy is determined by measuring the length of tunnels in cm per stalk for 10 plants and is expressed as the average length (sd in brackets) of tunnels per maximum number of tunnels per plant. In the field, ECB efficacy is expressed as the average of 3 values obtained for different groups of 10 plants.

TABLE 4 ECB efficacy trials in the greenhouse and in different locations in the field Greenhouse Field 1 Field 2 ECB efficacy ECB efficacy ECB Efficacy p35S-cry1Ab CE024-1301 1.2 (1.47)/2 0.4 (0.53)/4 0.05 (0.07)/1 PGos-cab- cry1Ab CE050-0802 0 (0)/0 0 (0)/0 0 (0)/14 CE052-0101 0.6 (0.84)/2 0.37 (0.43)/3 0.17 (0.23)/2 CE052-0205 1.6 (2.4)/6 0.08 (0.072)/1 0.05 (0.07)/1 CE053-0605 0.4 (0.69)/2 0.113 (0.1)/1 0.047 (0.08)/1 0.09 (0.085)/1 0 (0)/0 CE053-0702 0.6 (0.96)/2 0.4 (0.53)/5 0.055 (0.097)1 CE057-0201 0.21 (0.26)/1 0.2 (0.35)/6 CE060-0402 0.7 (1.64)/5 0.91 (0.44)4 0.4 (0.692)/3 CE061-0301 0.7 (1.16)3 0.22 (0.39)/4 0.0625 (0.088)/1 CE061-1102 2.7 (1.76)/5 1 CE061-1402 2.8 (4.52)/8 0.22 (0.47)/2 CE061-1502 1.7 (0.97)/(3) 0.08 (0.14)/1 0 (0)/0 CE062-0201 0 (0)/0 0 (0)/0 CE067-0401 0 (0)/0 0.205 (0.113)1 CE068-0903 0.07 (0.11)/2 0.04 (0.0072)/1 pGos-gos- Cry1Ab CE180-0303 0 (0)/0 0 (0)/0 0 (0)/0 CE181-0201 0.1 (0.32)/1 0.36 (0.34)/2 0 (0)/0 CE182-0101 0 (0)/0 0.07 (0.1)/0 CE183-0501 0 (0)/0 00.056 (0.08)/1 CE183-0601 1 0 (0)/0 CE184-0101 0.7 (1.06)/3 0.51 (0.35)/2 0 (0)/0 CE184-0609 0 (0)/0 0 (0)/0 CE184-0801 0.11 (0.33)/1 0 (0)/0 0 (0)/0 CE186-0203 0 (0)/0 0.037 (0.064)/1 CE187-0302 0.08 (0.14)/2 0 (0)/0 CE187-0408 0.1 (0.32)/1 0 (0)/0 0.11 (0.19)1 CE187-0803 0.096 (0.17)/1 0.097 (0.16)1 CE193-1002 0.15 (0.17)/1 0.33 (0.58)/4 CE196-0201 0.4 (1.26)/0 0 (0)/0 0.144 (0.14)1 CE197-0102 0 (0)/0 CE198-0401 0.9 (1.28)/3 0.09 (0.08)/1 0 (0)/0 CE198-0802 0 (0)/0 0.17 (0.21)/3 0.03 (0.057)/1 CE198-1401 0 (0)/0 0.047 (0.0082)/1 0 (0)/0 CE198-1702 0.1 (0.32)/1 0.55 (0.74)/4 0 (0)/0 CE198-2101 0.1 (0.32)/1 0 (0)/0 0.085 (0.12)1 CE198-2501 0 (0)/0 0 (0)/0 0 (0)/0 PUbi-cry1Ab ACE054- 158.8 (32.3)/200 19.0 (1.4)30 00103 ACE054- 166.2 (31.2)/235 20.7 (0.98)/27 01502 ACE054- 114.5 (92.1)/270 22.4 (2.9)/42 02803 B73 control 150.6 (40.3)/211 25.7 (4.8)/39 27.6 (7.4)/62

The p35S-cry1Ab event gave absolute ECB control, both in the greenhouse as in field-trials at different locations which correlated with the high-dose expression (as described above). Similarly, the pGos-gos-cry1Ab and PGos-cab-cry1Ab events displayed good ECB control at all locations tested. The pUbi-cry1Ab event did not show good ECB control.

No phytotoxicity was observed for any of the p35S-cry1Ab or pGos-cab-cry1Ab events. Selfed seed of pGos-gos-cry1Ab events showed segregation in the field of normal green plants and stunted, yellowish plants, from which the lower leaves are dying, suggestive of some impact on agronomic performance.

Fourteen events obtained with the pTR2′-cry1Ab construct were evaluated for ECB efficacy in the greenhouse and in field trials (Table 5). Four of the five single-copy events (indicated with an asterisk) gave total ECB2 control.

TABLE 5 ECB Efficacy in the Greenhouse and in the Field for pTR2′-cry1Ab events ECB efficacy ECB efficacy ECB efficacy Greenhouse Field Field Average (sd)/max average (sd)/max Average (sd)/max Event tunnels/pl tunnels/pl tunnels/pl WI602-0402 0.1 (0.31)/1 0.24 (0.41)/2 0.21 (0.01)/2 WI604-1602 0.2 (0.42)/1 0.05 (0.071)/1 WI606-0406* 51.3 (73.5)/195 8.41 (1.8)/32 7.45 (0.07)/30 WI606-0802 3.3 (2.6)/8 0 (0)/0 0.05 (0.07)/1 WI606-1206 0.38 (0.74)/2 0.17 (0.29)/3 0.1 (0.14)/1 WI600-0218 2.0 (1.87)/6 WI600-1402* 29.4 (45.8)/142 6.55 (2.47)/28 WI602-0202* 168.5 (26.9)/200 12.6 (3.8)/31 WI604-0604* 102.5 (83)/251 3.7 (2.0)/25 WI602-0802 0 (0)/0 0.33 (0.15)/3 WI602-0204 66 (68.2)/160 WI602-1002 102.4 (63.4)/215 WI606-0602 45.5 (52.8)/160 WI600-0802 0.9 (1.44)/4 0.06 (0.11)/2 0 (0)/0 B73 control 150.6 (40.3)/211 25.7 (4.8)/39 27.6 (7.4)/62

No penalty on agronomic performance was observed for the plants after second selfing (ear to row) of the different TR2′ events in any of the locations tested.

Efficacy Against Sesamia nonagrioides

Five mid whorl corn plants obtained with the pTR2′-cryl Ab construct were each infested with 2 egg masses. Damage was scored after 14 days and the number of larvae were counted. Damage ratings were averaged over the five plants. Non-transformed B73 plants were similarly infested as controls. Results are shown in Table 6.

TABLE 6 Number of Plant Cm tunnels Event larvae per plant height in cm per plant WI600-0802 0.6 (1.3) 198 (8.4) 0 B73 Control 197.2 (14.0) 84 (5.5) 70 (9.4)

Example 3

Cry1Ab Gene in Cotton

I. Development of Cry1Ab Events in Cotton

A construct was made for the expression of the modified cry1Ab gene in cotton. The pTSVH0203 construct contains the modified cry1Ab coding sequence (encoding part of the Cry1Ab5 protein described by Höfte et al.1986 having an insertion of an alanine codon (GCT) 3′ of the ATG start codon). The cry1Ab coding sequence is under control of the constitutive promoter 35S (Odell et al. 1985), linked to the leader sequence of the tapetum E1 gene from Oryza sativa (WO92/13956) with the 3′ ocs terminator (fragment containing polyadenylation signals from the 3′untranslated region of the octopine synthase gene from the TL-DNA of pTiAch5, De Greve et al., 1983). The construct additionally comprises the bar coding sequence (the coding sequence of phosphinothricin acetyl transferase of Streptomyces hygroscopicus; Thompson et al., 1987) under control of the 35S promoter.

PTSVH0203 P35S2-GE1-cry1Ab53-3′ocs<>p35S3- p35S-cry1Ab-ocs bar-3′nos This construct was used for transformation of cotton. The obtained events were subjected to molecular analysis to confirm presence of the transgene.

Analysis of Events

1. Expression of Modified Cry1Ab Protein

Expression of modified Cry1Ab protein was determined in leaf, terminal, square, flower and boll samples using ELISA (Table 7). Results represent percentage of Cry1Ab protein of total protein content as an average of samples taken from 9 plants. The time point at which samples were taken is represented as days after planting (in brackets).

TABLE 7 Cry1Ab expression as measured by ELISA in different tissues ELISA, Cry1Ab expression in %, 9 plants Leaf Leaf Leaf Terminal leaf Terminal leaf Terminal leaf Square Flower Bolls Events (60) (65–80) (115–120) (60) (65–80) (115–120) s (60) (65–80) (115–120) COCE040-04702A 0.024 0.008 0.005 0.005 0.012 0.006 0.002 0.003 0 COCE040-04702B 0.011 COCE040-3106- 0.017 0.022 0.012 0.015 0.011 0.007 0.003 1143 COCE040-3106- 0.018 1144

2. Laboratory Toxicity Assay

CBW larvae were fed on squares, leaf and terminals obtained from leaves in the field. Weight of surviving larvae was measured for the different events (Table 8). Samples from two non-transgenic plants and from one plant comprising a herbicide resistance gene (LL25 event) were used as control

TABLE 8 Toxicity of modified Cry1Ab expressed in cotton to CBW larvae Weight in mg of surviving larvae Event Squares Terminals Leaf  5 2.8 0 8.7  8 (- control LL25) 22.3 1.7 15.5 11 0 0 18 13 4.3 1 20.6 16 6 0.3 23 21 (- control non-transgenic) 16.5 8.7 26.3 23 (- control non-transgenic) 8.2 38.5 29

3. Efficacy of Insect Control in the Field

In the field, plants were artificially infested with neonate CBW larvae. Damage to leaves, terminals, squares, white bloom and bolls was rated and counts were made each 8 days in August and September. A mean was made over the five observations dates. Statistical analysis indicated that significant differences were found in the mean damage severity of terminals, white blooms and bolls. In addition, the mean number of damaged squares and damaged bolls was higher for the controls than for the Cry1Ab events; The mean number of larvae in squares was also significantly reduced compared to controls.

Example 4 Cry1Ab Gene in Rice

A construct was made for the expression of the modified cry1Ab gene in rice. The construct contains the modified cry1Ab coding sequence (encoding part of the Cry1Ab5 protein described by Höfte et al. 1986 having an insertion of an alanine codon (GCT) 3′ of the ATG start codon). In this construct, the cry1Ab coding sequence is under control of the promoter with the 5′ leader sequence of the GOS2 gene from rice, containing the second exon, the first intron and the first exon of the GOS transcript (de Pater et al., 1992). This construct was used for transformation of rice. The obtained events were subjected to molecular analysis to confirm presence of the transgene.

Plants of 25 different events were tested for control of rice leaf folder, yellow stem borer, and pink stem borer by counting the number of surviving larvae. Damage to the plants was assessed as damage to the leaves (for leaf folder) or the number of deadhearts per number of tillers at the preflowering stage (yellow stem borer and pink stem borer) or the number of whiteheads per number of tillers at the flowering stage (yellow stem borer). For only 2 events plants were found on which some (5–7 out of 25) of the larvae survived. All plants of all other events showed 90–100% dead larvae. While the control plants were either completely dried up or showed many folded leaves when infested with rice leaf folder, none of the plants of the cry1Ab events showed any significant damage. Similarly, no deadhearts or whiteheads were detected for the plants of the cry1Ab events, infested with yellow stem borer or pink stem borer.

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1. A DNA sequence comprising a coding region encoding a modified Cry1Ab protein, wherein the DNA sequence comprises the nucleotide sequence of SEQ ID NO:
 2. 2. An isolated chimeric gene comprising the DNA sequence of claim 1 under control of a plant-expressible promoter.
 3. A recombinant vector comprising the chimeric gene of claim
 2. 4. A transgenic plant cell comprising the chimeric gene of claim
 2. 5. A transgenic plant comprising the plant cell of claim
 4. 6. The transgenic plant of claim 5, which is corn, cotton, or rice.
 7. A seed of the plant of claim 6, wherein the seed comprises the DNA sequence of SEQ ID NO:
 2. 8. A method for protecting a plant from lepidopteran insects pests, wherein the method comprises introducing into the plant the chimeric gene of claim 2 in a manner such that the plant produces an insect-controlling amount of the modified Cry1Ab protein.
 9. A method for protecting a plant from Lepidopteran insects, comprising growing a plant in a field, wherein said plant produces an insect-controlling amount of a modified insecticidal crystal protein comprising the amino acid sequence of SEQ ID NO: 1 in cells of said plant, wherein the cells comprise a chimeric gene comprising the DNA sequence of SEQ ID NO: 2 under control of a plant-expressible promoter.
 10. The method of claim 9, wherein the cells comprise a chimeric gene comprising the DNA sequence of SEQ ID NO:2 under control of a plant-expressible promoter. 